Background A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10-12) in patients with secondary progressive multiple sclerosis. Conclusions An underlying low-grade chronic intravascular haemolysis is a potential source of the iron whose deposition along blood vessels in multiple sclerosis plaques contributes to the neurodegeneration and consequent brain atrophy seen in progressive disease. Chelators of free serum iron will be ineffective in preventing this neurodegeneration, because the iron (Fe2+) is chelated by haemoglobin.

This work is performed using the serum samples from the MS-STAT study which showed a small yet significant reduction in the rate of brain volume with high-dose (80mg) simvastatin. The aim of this study was to find potential biomarkers of brain atrophy.

Here, Lewin et al. report a new potential candidate; free serum haemoglobin. It does not come as news to me that this wasn't picked up by other seasoned proteomics researchers before, as it's easy to overlook proteins where there is lack of substantiating evidence in the disease under question. That is, this may be present in previous screens but not flagged as significant by the researchers reporting it! In the same way I'm surprised that neurofilament proteins were not flagged in Lewin's work as I know them to be a consistent finding in sample screens for progressive MS and increased with brain volume loss...

Proteomics also suffers from pre-analytical variations i.e. sample processing, storage etc., which may lead to differential findings from group to group. This is why, a confirmatory check is needed (i.e. the same change is demonstrated by another methodology, for example ELISA), which is what is performed here. They demonstrate that there is a similar rise in serum haemoglobin using commercial test (provided by Abcam). But they do not provide any validation data on this, which is a paramount requirement in this field of research. The European Biomarker Consortium provided a guidance document with regard to reporting on new biomarkers (Guidelines for uniform reporting of body fluid biomarker studies in neurologic disorders. Gnanapavan S, Hegen H, Khalil M, Hemmer B, Franciotta D, Hughes S, Hintzen R, Jeromin A, Havrdova E, Tumani H, Bertolotto A, Comabella M, Frederiksen J, Álvarez-Cermeño JC, Villar L, Galimberti D, Myhr KM, Dujmovic I, Fazekas F, Ionete C, Menge T, Kuhle J, Keir G, Deisenhammer F, Teunissen C, Giovannoni G), to avoid scientists spending time on researching random findings! I suppose it's fine because the authors state that "these results do not suggest that free serum haemoglobin concentration
is useful in the differential diagnosis of neurological disease", which is smart!

Let's look at the hypothesis, this is an interesting one and well worth taking a second look. We know that in all neurodegenerative disorders, including MS, there is an increase in the iron deposition in the brain. Normally, there is iron bound to haemoglobin in red blood cells. When you spin blood to obtain the serum the red blood cells (which are heavier) separate out at the bottom of the tube. Therefore, any free blood in the sample, outside of rubbish venepuncture technique (I'm assuming that haemolysed samples were excluded prior to analysis, as this is a requirement for proteomic studies), is indicative of blood break down (or haemolysis). There is then a possibility, that this may cause problems (see above figure for a potential mechanism); although I don't think this is a direct causal evidence for brain atrophy (i.e. may be epiphenomena), as autoimmune haemolytic anaemia (blood disorder which is both genetic and caused by other illnesses) has no reported mention of brain volume loss. Clearly, more work is needed in this area. Moreover, other groups looking at this also need to examine haptoglobin levels, as haptoglobin binds free serum haemoglobin and genetic variations in haptoglobin between individuals has been reported to affect free serum haemoglobin levels.

Finally, there is no observed treatment effect on free serum haemoglobin of high-dose simvastatin. Lewin et al. state "This effect was independent of the beneficial treatment effect of
simvastatin, because there was no association between free haemoglobin
concentration and simvastatin treatment", they also state "The results presented here show that a rise in the concentration of free
haemoglobin in the serum was associated with the rate of brain atrophy
in this cohort of patients with SPMS". Are they, therefore, implying that high-dose simvastatin does not in fact lower the likelihood of brain atrophy! Maybe, I'm putting words in their mouth!! Please place me on a direct line to a good statistician!

4 comments:

When I read this study last month I was actually wondering what would make the Blood Brain Barrier become so weak? And would there be any relationship between circulating red blood cells and T and B cell levels?

Hi Cinara, the blood brain barrier is leaky in MS from inflammatory cytokines making it more permeable to allow WBC to transfer across. RBC are in fact much smaller than WBC in size ratio but unlike the WBC can't deform their cytoskeleton to squeeze through, they'd simply pop. So there isn't a direct relationship between the two. This paper claims that there is lysis of RBC occurring in the circulation in MS which will need to be investigated in further details. Healthy RBC don't lyse there needs to be something going on in the vessels to cause it. But as you can see from my article that the work needs to be interpreted with caution. It may be occurring alongside another process which hasn't been adequately captured in MS as yet (i.e. an epiphenomenon).

The flow dynamics in vessels are such that RBCs rarely flow in the periphery. If there are fibrin strands blocking the vessels then they can be sliced as they pass through - this is the commonest cause of acquired haemolysis (or red cell lysis).

PML Risk Infographic

Search this Blog

Follow by Email

Subscribe To

Translate

Disclaimer

General Disclaimer: Please note that the opinions expressed here are those of the individual bloggers and do not necessarily reflect the positions of the Barts and The London School of Medicine and Dentistry, Queen Mary University of London or Barts Health NHS Trust.

Survey Disclaimer: No personal identifiers will be collected as part of these surveys. By completing these surveys you are consenting to the data you provide being analysed by Professors Giovannoni and Baker and their collaborators. Results of these surveys will be presented on this blog and may be submitted for publication.